All eukaryotic cells are regulated by the phosphoinositide signal transduction cycle. In the phosphoinositide cycle, PI is phosphorylated on the fourth hydroxyl of the myo-inositol ring and then by the phosphatidylinositol-phosphate kinases (PIPK), forming phosphatidylinositol-4,5-bisphosphate (PIP2). PIP2 is essential as a precursor to many phosphatidylinositol-derived second messengers. By immunofluorescence, the PIPKIalpha is the only type I isoform present in nuclei at "nuclear speckles," and PIP2 is generated at these same sites and regulates speckle remodeling. Inositol-l,4,5-trisphsphate (IP3) kinases target identically with the PIPKIalpha, and the resulting inositol phosphates generated may also impact the targeting or activities of mRNA processing enzymes. The phosphorylation of IP3 by the IPn kinases results in synthesis of IP6, which is a substrate for the IP6 kinase. The type I IP6 kinase regulated the G2/M cell cycle transition. The broad and long-term goal of this proposal is to define the underlying mechanisms by which the PIPKIn, IP3, and IP6 kinases function in nuclear signaling pathways. The proposed work will critically assess this hypothesis with the following Specific Aims: (1) Investigate the role of PIPKIalpha in nuclear signaling. The nuclear PI4,5P2 pathway will be manipulated to knockdown nuclear PI4,5P2 content. The cellular phenotypes will be defined by assessing speckle remodeling, mRNA processing and export, cell cycle, and apoptosis. PIPK( interactions with nuclear proteins will reveal the underlying molecular mechanisms; (2) Define the role of nuclear IP3 kinases. Phosphorylation of IP3 initiates the synthesis of the higher inositol-phosphate messengers. Two mammalian IP3 kinases are nuclear at speckles and may utilize the IP3 generated from PI4,5P2 hydrolysis. The IP3 kinase functions will be defined by siRNA ablation of expression, and the resulting role of IP n generation and cellular phenotypes will be characterized; (3) Define the role of the type IIPs kinase in G2/M cell cycle progression. The IP6 kinases phosphorylate IPs and IP6 to PPIP4 and PPIPs. Cells with the type I IP6 kinase knocked out show a dramatic block in G2/M cell cycle progression. This phenotype will be characterized, and the G2/M signaling pathways disrupted will be investigated. A role for PIPKIalpha and IPN kinases in mRNA metabolism, cell growth regulation, or DNA repair has many implications for nuclear signal transduction in general and specifically for proliferative diseases and cancer.